1. Field of the Invention
The present invention relates to purification and chemical modification of proteins to prolong their circulating lifetimes and reduce their immunogenicity. More specifically, the invention relates to the removal of aggregates larger than octamers from urate oxidases (uricases) prior to conjugation of poly(ethylene glycols) or poly(ethylene oxides). This substantially eliminates uricase immunogenicity without compromising its uricolytic activity.
2. Description of the Related Art
Statements contained in this background section do not constitute an admission of prior art, but instead reflect the inventors"" own subjective comments on and interpretations of the state of the art at the time the invention was made. These interpretations may include personal, heretofore undisclosed, insights of the inventors, which insights were not themselves part of the prior art.
Urate oxidases (uricases; E.C. 1.7.3.3) are enzymes that catalyze the oxidation of uric acid to a more soluble product, allantoin, a purine metabolite that is more readily excreted. Humans do not produce enzymatically active uricase, as a result of several mutations in the gene for uricase acquired during the evolution of higher primates. Wu, X, et al., (1992) J Mol Evol 34:78-84. As a consequence, in susceptible individuals, excessive concentrations of uric acid in the blood (hyperuricemia) and in the urine (hyperuricosuria) can lead to painful arthritis (gout), disfiguring urate deposits (tophi) and renal failure. In some affected individuals, available drugs such as allopurinol (an inhibitor of uric acid synthesis) produce treatment-limiting adverse effects or do not relieve these conditions adequately. Hande, K R, et al., (1984) Am J Med 76:4-56; Fam, A G, (1990) Baillixc3xa8re""s Clin Rheumatol 4:177-192. Injections of uricase can decrease hyperuricemia and hyperuricosuria, at least transiently. Since uricase is a foreign protein in humans, however, even the first injection of the unmodified protein from Aspergillus flavus has induced anaphylactic reactions in several percent of treated patients (Pui, C-H, et al., (1997) Leukemia 11:1813-1816), and immunologic responses limit its utility for chronic or intermittent treatment. Donadio, D, et al., (1981) Nouv Presse Mxc3xa9d 10:711-712; Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic 50:553-554.
U.S. patent application Ser. No. 09/370,084 and published International Application No. PCT/US99/17514, the entire contents of which are incorporated herein by reference, disclose poly (ethylene glycol)-urate oxidase (PEG-uricase) that retains at least about 75% of the uricolytic activity of unconjugated uricase and has substantially reduced immunogenicity. In one such purified uricase, each subunit is covalently linked to an average of 2 to 10 strands of PEG, wherein each molecule of PEG may have a molecular weight between about 5 kDa and 100 kDa.
The aggregation of proteins is known to increase their immunogenicity. This understanding has contributed to the development of methods for intentionally aggregating proteins by treatments such as thermal denaturation and cross-linking by exposure to glutaraldehyde prior to use in the preparation of vaccines or for immunization of animals to produce antisera.
Unintentional aggregation of proteins has also been recognized as contributing to immunization or sensitization during clinical use of therapeutic proteins, e.g. for human gamma globulin (Henney et al. (1968) N. Engl. J Med. 278:2244-2246) and for human growth hormone (Moore et al. (1980) J Clin. Endocrinol. Metab. 51:691-697). The contribution of aggregates to the immunogenicity of human interferon alpha has been demonstrated in BALB/c mice (Braun et al. (1997) Pharm. Res. 14:1472-1478) and an enzyme-linked immunosorbent assay (ELISA) has been developed for their measurement (Braun et al. (1997) Pharm. Res. 14:1394-1400).
In contrast to the known effects of aggregation on the immunogenicity of proteins, there are not reports of the effect of aggregation on the immunogenicity of proteins conjugated to poly(alkylene glycols) such as PEG. There is a need for poly(alkylene glycol)-uricase conjugates that substantially eliminates uricase immunogenicity without compromising its uricolytic activity. The present invention provide such compositions.
Conjugation of proteins with poly(alkylene glycols), especially PEG, produces conjugates with reduced immunogenicity and increased persistence in the bloodstream. In attempting to produce substantially non-immunogenic conjugates of uricase that retain substantially all of the uricolytic activity of the unmodified uricase preparation, it was discovered that traces of large aggregates of uricase in the starting material were surprisingly effective at provoking both antibody formation and accelerated clearance from the circulation, both of which are deleterious, after repeated injections of PEG conjugates prepared from uricase containing such aggregates. Surprisingly, the present inventors found that the increased immunogenicity and accelerated clearance were not due to the presence of well-defined, moderate-sized aggregates of the uricase subunit that are larger than the native tetramer, e.g. aggregates containing eight subunits (octamers). The octameric form of uricase is present at sufficiently high concentrations in most preparations of uricase to be detectable by its absorbance of UV light, e.g. at 214 nm or 276 nm, or by its contribution to the refractive index or other measurements of protein concentration. Nevertheless, the octamers themselves were found to contribute minimally to the immunogenicity and accelerated clearance of PEG-uricase conjugates, in contrast with the much smaller quantities of the much larger aggregates that are undetectable by UV absorbance under the conditions tested but are readily detected by static (Raleigh) or dynamic light scattering. Therefore, the removal of such traces of very large aggregates prior to conjugation with PEG was found to decrease the immunogenicity and the accelerated clearance of the resultant PEG-uricase conjugates to a surprising extent.
One embodiment of the present invention is purified urate oxidase (uricase) substantially free of aggregates larger than octamers. Preferably, the uricase is mammalian uricase. More preferably, the uricase is porcine liver, bovine liver or ovine liver uricase. In one aspect of this preferred embodiment, the uricase is recombinant. In another aspect of this preferred embodiment, the uricase has substantially the sequence of porcine, bovine, ovine or baboon liver uricase. Advantageously, the uricase is chimeric. Preferably, the uricase is PKS uricase. In another aspect of this preferred embodiment, the uricase has substantially the sequence of baboon liver uricase in which tyrosine 97 has been replace by histidine. Preferably, the uricase comprises an amino terminus and a carboxy terminus, and wherein the uricase is truncated at one terminus or both termini. Advantageously, the uricase is a fungal or microbial uricase. Preferably, the fungal or microbial uricase is isolated from Aspergillus flavus, Arthrobacter globiformis, Bacillus sp. or Candida utilis, or is a recombinant enzyme having substantially the sequence of one of said uricases. Alternatively, the uricase is an invertebrate uricase. Preferably, the invertebrate uricase is isolated from Drosophila melanoguster or Drosophila pseudoobscura, or is a recombinant enzyme having substantially the sequence of one of said uricases. In another aspect of this preferred embodiment, the uricase is a plant uricase. Preferably, the plant uricase is isolated from root nodules of Glycine max or is a recombinant enzyme having substantially the sequence of the uricase.
In one aspect of this preferred embodiment, the uricase described above is conjugated to poly(ethylene glycol) or poly(ethylene oxide), under conditions such that the uricase in the conjugate is substantially free of aggregates larger than octamers. Preferably, the uricase is conjugated to poly(ethylene glycol) or poly(ethylene oxide) via a urethane (carbamate), secondary amine or amide linkage. In one aspect of this preferred embodiment, the poly(ethylene glycol) is monomethoxy poly(ethylene glycol). In another aspect of this preferred embodiment, the poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight between about 5 kDa and 30 kDa. Preferably, the poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight between about 10 kDa and 20 kDa. Advantageously, the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 2 and 12 per uricase subunit. More advantageously, the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 6 and 10 per uricase subunit. Most advantageously, the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 7 and 9 per uricase subunit. Preferably, the poly(ethylene glycol) or poly(ethylene oxide) is linear. Alternatively, the poly(ethylene glycol) or poly(ethylene oxide) is branched.
The present invention also provides a pharmaceutical composition for lowering uric acid levels in a body fluid or tissue, comprising the uricase conjugate described above and a pharmaceutically acceptable carrier. Preferably, the composition is stabilized by lyophilization and dissolves upon reconstitution to provide solutions suitable for parenteral administration.
Another embodiment of the invention is a method for purifying uricase having reduced immunogenicity, comprising the step of separating uricase aggregates larger than octamers in uricase fractions, and excluding such aggregates from the purified uricase. Preferably, the separating step comprises the step of detecting aggregates larger than octamers from at least a portion of the uricase fractions and excluding the fractions containing the aggregates. Advantageously, the detecting step comprises measurement of light scattering.
The present invention also provides isolated uricase prepared by the method described above.